The aim of the present study was to purify the crude pectin extract obtained from gabiroba pulp, elucidate its chemical structure, and evaluate the antitumor potential of crude and purified pectin in a human glioblastoma model.
Carbohydrate Polymers 224 (2019) 115140 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Cytotoxic effect of crude and purified pectins from Campomanesia xanthocarpa Berg on human glioblastoma cells T Sarah da Costa Amarala,1, Shayla Fernanda Barbieria,1, Andrea Caroline Ruthesd,e, ⁎ Juliana Müller Barka, Sheila Maria Brochado Winnischofera,b,c, Joana Léa Meira Silveiraa,b, a Postgraduate Program in Biochemistry Sciences, Sector of Biological Sciences, Federal University of Paraná, Curitiba, PR, 81531-990, Brazil Department of Biochemistry and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil c Postgraduate Program in Cellular and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil d Division of Glycoscience, Royal Institute of Technology - KTH, Sweden e Department of Entomology and Nematology, University of Florida, Gulf Coast Research and Education Center (GCREC-UF), Wimauma, USA b ARTICLE INFO ABSTRACT Keywords: Gabiroba Pectin NMR analysis Glioblastoma cells Cytotoxicity ROS A new source of pectin with a cytotoxic effect on glioblastoma cells is presented A homogeneous GWP-FP-S fraction (Mw of 29,170 g mol−1) was obtained by fractionating the crude pectin extract (GW) from Campomanesia xanthocarpa pulp According to the monosaccharide composition, the GWP-FP-S was composed of galacturonic acid (58.8%), arabinose (28.5%), galactose (11.3%) and rhamnose (1.1%), comprising 57.7% of homogalacturonans (HG) and 42.0% of type I rhamnogalacturonans (RG-I) These structures were characterized by chromatographic and spectroscopic methods; GW and GWP-FP-S fractions were evaluated by MTT and crystal violet assays for their cytotoxic effects Both fractions induced cytotoxicity (15.55–37.65%) with concomitant increase in the cellular ROS levels in human glioblastoma cells at 25–400 μg mL−1, after 48 h of treatment, whereas no cytotoxicity was observed for normal NIH 3T3 cells This is the first report of in vitro bioactivity and the first investigation of the antitumor potential of gabiroba pectins Introduction Glioblastoma is one of the most frequent and most lethal malignant primary brain tumors and is associated with poor prognosis (Bailey et al., 2015) Current therapy includes surgical resection, followed by radiotherapy and/or concomitant adjuvant temozolomide (TMZ) chemotherapy (Arbab et al., 2017) Despite modern advance in therapies, over 90% of patients experience tumor recurrence, and the average period of survival for patients diagnosed with glioblastoma is only about 14 months (Bailey et al., 2015; Tanaka, Louis, Curry, Batchelor, & Dietrich, 2013) Therefore, the search for additional therapeutic strategies remains a high priority Recently, polysaccharides (such as pectins) have been exploited for their anticancer potential, due to their broad spectrum of therapeutic properties and their low toxicity to healthy cells (Munarin et al., 2015; Noreen et al., 2017) Although studies have shown that pectins are effective against a range of tumor models, including prostate cancer (Prado et al., 2017), breast cancer (Cobs-Rosas, Concha-Olmos, Weinstein-Oppenheimer, & Zúñiga-Hansena, 2015) and melanoma (Vayssade et al., 2010), most studies have focused on a colon cancer model (Zhang, Xu, & Zhang, 2015) To our knowledge, no report has been made on the antitumor potential of pectins against glioblastoma cells Pectins are anionic polysaccharides composed of (1, 4)-linked-Dgalacturonic acid residues and a variety of neutral monosaccharides, such as rhamnose, galactose, and arabinose They can be classified into three main types, according to common features: Homogalacturonans (HG), type I Rhamnogalaturonans (RG-I), and type II Rhamnogalaturonans (RG-II) (Caffal & Mohnen, 2009; Chan, Choo, Young, & Loh, 2017; Yapo, 2011) These polysaccharides are the main component of the peels and pulps of several fruits, although most biological studies are performed with commercial pectins that have been extracted from citrus peel and apple pomace (Noreen et al., 2017; Zhang et al., 2015) As a result, a large number of native fruits around the world continue to go underused, despite the abundance of the species Brazil is believed to harbor the earth's richest flora, including several native fruits from the Myrtaceae family (Donado-Pestana et al., 2018) Corresponding author at: Department of Biochemistry and Molecular Biology, P.B.19046, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil E-mail address: jlms12@ufpr.br (J.L.M Silveira) These authors equally contributed to this work ⁎ https://doi.org/10.1016/j.carbpol.2019.115140 Received 16 April 2019; Received in revised form 26 July 2019; Accepted 27 July 2019 Available online 02 August 2019 0144-8617/ © 2019 Elsevier Ltd All rights reserved Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al Biological properties have been reported of polysaccharides extracted from Myrtaceae family fruits A pectic arabinogalactan from edible jambo (Syzygium jambos) fruits showed immunomodulatory properties (Tamiello, Nascimento, Iacomini, & Cordeiro, 2018), while water-extracted polysaccharides from guava (Psidium guajava and Psidium littorale) fruits exhibited anti-glycated and α-glucosidase inhibitory activity (Yan, Lee, Kong, & Zhang, 2013; Zhang et al., 2016) This work investigated a Brazilian Myrtaceae family species, Campomanesia xanthocarpa Berg, popularly known as gabiroba (Barroso & Barroso, 1978) This species is used in folk medicine to treat such pathologies as inflammatory, renal, and digestive diseases; obesity; and hypercholesterolemia (Alice et al., 1995) In addition, scientific studies have confirmed that the leaf and fruit extracts of C xanthocarpa demonstrate a broad spectrum of therapeutic effects, including: antioxidant properties (Pereira et al., 2012), antibacterial effects (Czaikoski, Mesomo, Krüger, Queiroga, & Corazza, 2015), antiulcerogenic effects (Markman, Bacchi, & Kato, 2004), antidiabetic effects (Vinagre et al., 2010), reduction of cholesterol levels (Viecili et al., 2014) and obesity (Biavatti et al., 2004) However, most previous studies focused only on low molar mass compounds, and the aforementioned health benefits were attributed to the presence of vitamins (mainly ascorbic acid), flavonoids, volatile oils, carotenoids, and phenolic compounds In our previous works, polysaccharides extracted from gabiroba pulp were analyzed (Barbieri et al., 2017), and the presence of pectins was demonstrated (Barbieri et al., 2019) However, the bioactivities of pectins extracted from gabiroba fruits, as well as their antitumor potential, have remained unexplored Thus, the aim of the present study was to purify the crude pectin extract obtained from gabiroba pulp, elucidate its chemical structure, and evaluate the antitumor potential of crude and purified pectin in a human glioblastoma model was deionized with cation exchange resin and dialyzed with Cellulose 6–8 kDa cut off membrane (Spectrum Labs™), where the precipitation of some of the material could be observed inside the membrane Thus, the sample was removed from the membrane and centrifuged (3000 rpm, 20 min, °C), resulting in an insoluble fraction (GWP-FP-P) and a soluble fraction (GWP-FP-S) The soluble fraction GWP-FP-S was freezedried, structurally characterized, and analyzed for the biological activity tests (Fig 1) 2.2 Monosaccharide composition Uronic acid content of the GWP-FP-S fraction was quantified by the colorimetric m-hydroxybiphenyl method, using galacturonic acid as the standard (Blumenkrantz & Asboe-Hansen, 1973) The identity of the uronic acid was determined by anion exchange chromatography, with pulse amperometric detection (HPAEC-PAD) Samples were hydrolyzed with mol L−1 TFA for h at 100 °C, dried, and washed with methanol (x 3) to remove the acid Hydrolyzed samples (1 mg mL−1) were injected into a Thermo Scientific Dionex ICS-5000 chromatograph (Thermo Fisher Scientific, USA) with CarboPac PA20 column (3 × 150 mm), using a gradient of 0.5 mol L−1 NaOH and mol L−1 NaOAc as eluent (Nagel, Sirisakulwat, Carle, & Neidhart, 2014) in an N2 atmosphere in a flow of 0.2 ml min−1 at 24 °C The analysis was carried out in triplicate, and the data were collected and analyzed using the Chromeleon TM 7.2 Chromatography Data System software The neutral monosaccharides were evaluated through total acid hydrolysis, with mol L−1 TFA for h at 100 °C The hydrolysates were converted to alditol acetates through treatment with NaBH4 (Wolfrom & Thompson, 1963a), followed by acetylation with acetic anhydride (Ac2O)-pyridine (1:1, v/v, mL) at 100 °C for 30 (Wolfrom & Thompson, 1963b) The alditol acetates were extracted with CHCl3 and were analyzed in a Thermo Scientific Trace GC Ultra gas chromatograph, with a mixture of He, N2, and compressed air as the carrier gas at ml min−1 The chromatograph also used a DB-225-MS column (0.32 mm internal diameter x 30 m x film thickness 0.25 μm) programmed from 100 °C to 230 °C at a heating rate of 60 °C min−1 The alditol acetates were identified by their profile, and retention times were compared with standards Materials and methods 2.1 Isolation of pectic polysaccharides Gabiroba fruits were collected and prepared according to Barbieri et al (2017) Briefly, crude pectic fraction from gabiroba pulp was obtained by hot water extraction (GW) as reported in our previous work (Barbieri et al., 2019) In order to obtain a homogeneous fraction, GW was submitted to the fractionation process (Fig 1) During this process, the resulted freezing-thawing precipitated fraction (GWP) was treated with Fehling solution (Jones & Stoodley, 1965; Ruthes, Smiderle, & Iacomini, 2015) obtaining the precipitated fraction (GWP-FP), which 2.3 High performance size exclusion chromatography coupled to multidetectors (HPSEC-MALLS-RI) The homogeneity and average molar mass (Mw) of soluble polysaccharides were evaluated by high performance size exclusion chromatography (HPSEC), coupled with multi-angle laser light scattering (MALLS) (DSP-F, Wyatt Technology, Santa Barbara, CA, USA) and refractive index (RI) detectors (Waters 2410, Milford, MA, USA) (HPSECMALLS-RI) The log plot Mw versus elution time was calculated from the Rayleigh-Debye-Gans equation (Wyatt, 1993; Zimm, 1948) The chromatography was carried out on a Waters system containing four gel permeation columns packed with Ultrahydrogel® 2000, 500, 250, and 120, connected in series, with exclusion limits of × 106, × 105, × 104, and × 103 g mol−1, respectively The flow rate used was 0.6 ml min−1, with 0.1 mol L−1 sodium nitrite as the mobile phase and 0.2 g L−1 sodium azide as a preservative, at a temperature of 25 °C The data was collected and processed by Wyatt Technology ASTRA software, version 4.70.07 2.4 Protein quantification Protein content of the GWP-FP-S fraction was determined using the Bradford method (Bradford, 1976) A calibration curve of bovine serum albumin was built as a standard, and the results were expressed in g protein/100 g of sample Fig Scheme of fractionation of water-extracted polysaccharides from gabiroba pulp (Campomanesia xanthocarpa) *GW and GWP-TEP was previously characterized (Barbieri et al., 2019) Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al 2.5 Nuclear magnetic resonance (NMR) spectroscopy permeable probe, which oxidizes intracellularly to the highly fluorescent DCF (2′,7′-dichlorofluorescein) (Kalyanaraman et al., 2012) U251-MG and T98 G glioblastoma cells (4 × 103 cells/well) were seeded on black 96-well plates for 24 h of incubation, and treated as previously described for the cytotoxicity assay Subsequently, the culture medium was removed, cells were washed with PBS, and then incubated with mM of DCFH-DA at 37 °C for 30 in the dark Fluorescence was measured on a spectrofluorometer (Infinite M200, Tecan Trading AG, Switzerland) with a 485 nm excitation filter and a 520 nm emission Hydrogen peroxide (H2O2, 400 μM) was used as a positive control 30 before the measurement Values were normalized using crystal violet assay standardization and the intracellular fluorescence levels of control cells (vehicle used to dissolve the extract milliQ water) were considered as Mono-dimensional (13C- and 1H-) and bi-dimensional (1H/13C HSQC) NMR spectra were acquired at 70 °C on a Bruker AVANCE III 400 NMR spectrometer and equipped with a 5-mm multinuclear inverse detection probe with z-gradient, operating at 9.5 T and observing 1H at 400.13 MHz and 13C at 100.61 MHz The samples were acquired in D2O with chemical shifts expressed as δ (ppm), using the resonances of CH3e groups of acetone (1H at δ 2.22; 13C at δ 30.20) as internal references The data was collected and processed on Topspin 3.2 (Bruker BioSpin Corporation, Billerica, MA, USA) 2.6 Cell culture and treatment with pectic fractions U251-MG and T98 G human glioblastoma cell lines and normal murine fibroblast NIH 3T3 cell line were kindly provided by Dr Mari Cleide Sogayar, Cell and Molecular Therapy Center (NUCEL/NETCEM), Faculty of Medicine, University of São Paulo (FMUSP) Cells were cultured in a DMEM high-glucose medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS, Gibco) and 50 μg mL−1 gentamicin (Sigma-Aldrich), and maintained at 37 °C in a 5% CO2 atmosphere For the following experiments, GW and GWP-FP-S fractions obtained from gabiroba pulp were separately dissolved in ultrapure water − (5 mg mL 1) and stored at −20 °C until utilized 2.9 Statistical analysis The statistical analyses are expressed as mean ± SD of at least three independent experiments in quadruplicate Analyses were performed on GraphPadPrism software version 5.01 for Windows (GraphPad Software, San Diego, CA, USA), using a one-way analysis of variance (ANOVA) followed by Tukey’s A difference of p < 0.05 was considered statistically significant Results and discussion 2.7 Cytotoxicity assay 3.1 Structural characterization of purified fraction extracted from gabiroba pulp First, × 103 cells/well were seeded in 96-well culture plates for 24 h of incubation Then, cells were treated either with vehicle control (Ultrapure water – 18.2 MΩ cm−1, pH 6.9) or with different concentrations (10–400 μg mL−1) of GW and GWP-FP-S fractions The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method involves metabolically active cells reducing tetrazolium salt to formazan (Berridge, Herst, & Tan, 2005; Mosmann, 1983) For the MTT assay (Mosmann, 1983), cells were treated either with vehicle control or with 10, 25, 50, 100, 200, 400 μg mL−1 of GW and GWP-FP-S fractions Then, the culture medium was removed after 48 h of treatment and replaced with fresh medium containing 0.5 mg mL−1 MTT (Sigma- Aldrich); this was protected from light for h at 37 °C in a 5% CO2 atmosphere The medium was removed, and formazan production on viable cells was solubilized in dimethyl sulfoxide (DMSO) Absorbance was measured by spectrophotometry (Epoch, BioTek, USA) at 545 nm, and results were expressed as a percentage of control (vehicle), assigned as 100% of metabolically active cells Crystal violet is an alkaline dye with high affinity to nucleic acids extensively used to determine the number of cells through DNA staining of adhered and fixed cells (Kueng, Silber, & Eppenberger, 1989) For the crystal violet assay (Kueng et al., 1989), cells were treated either with vehicle control or with 25, 100, 400 μg mL−1 of GW and GWP-FP-S fractions After 48 h of treatment the culture medium was removed and washed with PBS Cells were then fixed in methanol for 10 min, stained with crystal violet for min, and then washed with PBS Afterwards, the stain was diluted in sodium citrate and quantified in a spectrophotometer (Epoch, BioTek, USA) at 545 nm Absorbance was proportional to cell number Results were calculated by comparing absorbance of treated-to-untreated cells (with control as the vehicle used to dissolve the fractions – Ultrapure water), assigning control absorbance as 100% of adhered cells Cell images were obtained using an inverted microscope (Axiovert 40, Carl Zeiss) GW fraction (5.06% yield), recently characterized by Barbieri et al (2019), was submitted to fractionation by freeze-thawing and the Fehling’s solution treatment, giving rise to a GWP-FP fraction (1.63% yield) This fraction was purified to give the fraction of interest, GWPFP-S (0.64% yield) (Fig 1) Fig 2A presents the HPSEC-RI elution profiles of the GWP-FP and GWP-FP-S fractions The GWP-FP fraction presented three peaks that eluted at retention times between 42 and 60 min, showing a heterogeneous profile, such as the GW fraction analyzed by Barbieri et al (2019); meanwhile, the GWP-FP-S fraction presented a homogeneous profile, with a single elution peak at 51 min, demonstrating that the polysaccharide fractionation process was efficient Assuming that achieve the homogeneity is the primordial step for studies of polysaccharide structure, pharmacology, and its structure-activity relationships (Shi, 2016), the GWP-FP-S was suitable for the investigation of its cytotoxic potential HPSEC-MALLS-RI chromatogram and molar mass distribution of the GWP-FP-S fraction were performed (Fig 2B) Through the light scattering signal (90°), a major peak can be observed between 35 and 45 (with an invisible signal to the RI detector), corresponding to its very low concentration A minor peak between 45 and 55 detected by light scattering (90°) was demonstrated as a major peak by the RI detector, showing a high concentration corresponding to the GWP-FP-S fraction The average molar mass (Mw) for GWP-FP-S was determined to be 29,170 g mol−1 (dn/dc 0.113) GWP-FP-S was predominantly composed of galacturonic acid (GalA, 58.8%), followed by arabinose (Ara, 28.5%), galactose (Gal, 11.3%), and rhamnose (Rha, 1.1%), suggesting the presence of pectins (Table 1) In comparison to the crude fraction GW (54.5% Ara; 33.5% GalA; 7.6% Gal) (Barbieri et al., 2019), the purified fraction GWP-FP-S, exhibited an increase in GalA and Gal followed by a decrease in Ara content (Table 1) Nevertheless, the content of GalA from the GWP-FP-S fraction was similar to that found by Maxwell et al (2016) for a modified sugar beet pectin (52 5% of GalA), which induced apoptosis of colon cancer cells The relative amount of HG and RG-I domains of the pectins was 2.8 Detection of intracellular reactive oxygen species (ROS) levels The intracellular levels of ROS were measured using DCFH-DA (2′, 7′-Dichlorofluorescin diacetate, Sigma-Aldrich) a non-fluorescent cell Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al Fig HPSEC-MALLS-RI elution profile (A) Elution profile by RI of the GWP-FP and GWP-FP-S fractions obtained from the gabiroba pulp (B) GWP-FP-S Fraction Molar mass distribution (MwD), light scattering (LS-90°), and refractive index (RI) estimated from the monosaccharide composition according to M’sakni et al (2006), using the equations: signal was not observed for GWP-FP-S A de-esterification may have occurred due to the use of alkaline pH in the Fehling’s treatment Deesterification was also observed by Nascimento et al (2017) on sweet pepper (Capsicum annum) pectin fraction after treatment with Fehling’s solution The 13C-NMR spectrum also showed signals at δ 107.6, δ 107.2, 106.5, and 106.3 that are attributed to α-L-Araf (C-1) Signals for the → 4)-β-D-Galp-(1→units were observed at δ 104.3 (C-1), δ 72.0 (C-2), δ 73.4 (C-3), δ 77.6 (O-substituted C-4), δ 74.5 (C-5), and δ 60.8 (C-6) All assignments obtained by 13C-NMR were confirmed by the analysis of the 1H/13C heterocorrelated HSQC-NMR spectrum (Table 2, Fig 4) The highest intensity peaks in the spectrum were attributed to →4)-α-D-GalAp-(1→ units, confirming the presence of HG in the GWPFP-S fraction Signals with lower intensity were also observed and were attributed to unsubstituted →2)-α-L-Rhap-(1→ and substituted →2,4)α-L-Rhap-(1→ units from the backbone of type I rhamnogalacturonan Arabinose and galactose side chains were ascribed to signals of linear backbone of →5)-α-L-Araf-(1→ units, as well as branched →3,5)-α-LAraf-(1→ units In addition, the presence of galactans was observed at correlations of →4)-β-D-Galp(1→ units (Colodel et al., 2018; Klosterhoff et al., 2018; Tamiello et al., 2018) With the analysis of H/13C heterocorrelated HSQC-NMR, it was possible to propose that the purified GWP-FP-S fraction consists of a pectin formed predominantly by HG regions and containing branched RG-I inserts with side chains of arabinans, galactans, and possibly arabinogalactans HG (%) = GalA (%) – Rha (%) and, RG-I (%) = [GalA (%) – HG(%)] + Rha + Ara + Gal Thus, the GWP-FP-S chain is represented by 42.0% of RG-I and 57.7% of HG These results showed a decrease in the RG-I proportion and an increase in the HG proportion of GWP-FP-S in relation to the GW fraction, which presented 65.3% RG-I and 31.9% HG (Barbieri et al., 2019) Structural differences in the main chain can be attributed to the process of purification According to the literature, HG-rich chains form a complex with Cu2+ and precipitate after being treated with Fehling’s solution (Nascimento, Iacomini, & Cordeiro, 2017) In addition, in order to estimate the extension of neutral side chains attached to the RG-I (Houben, Jolie, Fraeye, Van Loey, & Hendrickx, 2011), the ratio (Gal + Ara)/Rha was calculated The value obtained for GWP-FP-S was 37.6 (Table 1) Despite the differences in the proportions of the HG and RG-I domains of GWP-FP-S and GW, it can be observed that the values obtained to the neutral side chains attached to the RG-I (37.6 for GWP-FP-S and 38.8 for GW) was similar for both fractions, demonstrating that the side chain extension was not affected by the fractionation process adopted Regarding the protein content, GWP-FP-S showed no significant amount when evaluated by the Bradford method The GWP-FP-S fraction was investigated by 13C-NMR The 13C-NMR spectrum (Fig 3) showed a typical chemical shift from HG observed at δ 99.7 and 70.3, which correspond to C-1 and C-5, respectively, of unesterified α-D-GalAp units The remaining assignments of D-GalAp ring carbons were seen at δ 78.1 (O-substituted C-4/H-4), δ 68.3 (C-3), and δ 68.0 (C-2) Signals of C-6 from unesterified α-D-GalAp units could be assigned as δ 171.9 In the literature, the signal of methyl groups linked to α-D-GalAp units can usually be observed at δ 52.8 ppm (Colodel, Vriesmann, & Petkowicz, 2018; Nascimento et al., 2017); however, this 3.2 Cytotoxic effect of crude and purified pectin fractions extracted from gabiroba pulp Fig shows the cytotoxic effect of GW and GWP-FP-S pectins on glioblastoma cell lines U251-MG and T98 G cells were exposed to different concentrations of pectins (25–400 μg mL−1) for 48 h, and cell cytotoxicity was determined by crystal violet assay Both of these fractions were able to reduce the number of adherent Table Monosaccharide composition of crude and purified pectin fractions extracted from gabiroba pulp Monosaccharide composition (%)a Fraction GWd GWP-FP-S a b c d GalAb Rha Ara Xyl Man Gal Glc HG (%)c RG-I (%)c 33.5 58.8 1.6 1.1 54.5 28.5 1.0 0.2 tr tr 7.6 11.3 0.9 tr 31.9 57.7 65.3 42.0 % of peak area of monosaccharide composition relative to the total peak area, determined by GLC Uronic acids, determined using the m-hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973), and identified by HPAEC-PAD HG = GalA – Rha ; RG-I = 2(Rha) + Ara + Gal (M’sakni et al., 2006); tr = trace GW from Barbieri at al (2019) (Gal+Ara) Rha 38.8 37.6 Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al Fig 13 C NMR spectrum of the GWP-FP-S fraction from the pulp of gabiroba fruits, obtained at 70 °C in D2O (chemical shifts are expressed in δ, ppm) glioblastoma cells (stained by crystal violet assay) (Fig.5) These effects were also evidenced by optical microscopy, which showed that both GW (Fig 5D) and GWP-FP-S (Fig 5E) treatments promote significant changes in U251-MG cell morphology beyond altering cell number, as compared to a vehicle-treated control group (Fig 5C) All tested concentrations of both GW and GWP-FP-S fractions were able to reduce the percentage of adherent U251-MG and T98 G cells, reaching a decrease of 18.04–33.74% and 20.05–37.65% of adherent U251-MG cells after GW and GWP-FP-S treatment, respectively, at different concentrations Similar effects were also achieved for T98 G glioblastoma cells, where GW and GWP-FP-S treatments were able to inhibit 15.78–27.32% and 25.48–35.55% of adherent T98 G cells, respectively, indicating that the cytotoxic effect of these pectins was not selective, and is independent of cell-line-specific characteristics In view of this, the next investigations were continued with only one glioblastoma cell line (U251-MG) The cytotoxic effects were also confirmed by MTT assay (Fig 6) When U251-MG cells were treated with GW and GWP-FP-S at high concentrations (100–400 μg mL−1), the percentage of metabolicallyactive U251-MG cells were reduced in an equal manner However, U251-MG cells seemed to respond differently when pectin fractions were used at low concentrations A decrease in metabolic U251-MG cell Table H and 13C NMR chemical shifts (δ, ppm) of GWP-FP-S fraction from the pulp of gabiroba Glycosil residues Nucleus →4)-α -D-GalAp-(1→ 13 →2)-α-L-Rhap-(1→ 13 →2,4)-α-L-Rhap-(1→ →5)-α-L-Araf-(1→ →3,5)-α-L-Araf-(1→ t-α-L-Araf-(1→ →4)-β-D-Galp-(1→ C H C H 13 C H 13 C H 13 C H 13 C H 13 C H Chemical shifts, δ (ppm) 99.7 5.10 99.1 5.22 99.1 5.22 107.7 5.08 106.6 5.23 107.6 5.17 104.3 4.61 68.0 3.78 76.7 4.08 – 68.5 4.01 – 78.1 4.49 – 70.3 5.01 – – – – 81.4 4.13 – 76.8 4.01 – 79.8 4.27 72.0 3.68 76.7 3.97 73.4 3.75 82.8 4.19 82.3 3.90 84.1 4.04 77.6 4.15 66.8 3.88 66.6 3.91 61.3 3.73 74.5 3.70 16.5 1.25 16.8 1.32 60.8 3.80 Acetone was used as internal standard (δ2.22/30.2), and the analysis was carried out at 70 °C Fig 1H/13C HSQC- NMR spectrum of the GWP-FP-S fraction from the pulp of gabiroba fruits, obtained at 70 °C in D2O (chemical shifts are expressed in δ, ppm) Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al Fig Cytotoxic effect of GW and GWP-FP-S pectins Glioblastoma cell lines: (A) U251-MG and (B) T98 G cells were treated with GW or GWP-FP-S at different concentrations (25–400 μg mL−1) for 48 h The percentage of adherent cells were evaluated by crystal violet assay The values represent the means ± SD of percentage of cells compared with the control (data represent at least three independent experiments, each in quadruplicate) *p < 0.05; **p < 0.01; ***p < 0.001 Morphology of U251-MG cells: (C) Control group (cells treated with vehicle) (D) Cells treated with GW at 400 μg mL−1 (E) Cells treated with GWPFP-S at 400 μg mL−1 Magnification 100 × GW fraction did not alter their cellular metabolic activity, likely in an attempt to maintain survival This differential effect was no longer observed when the treatment reached higher concentrations of either fraction It is important to note that, from a pharmacological point of view, cytotoxicity at lower concentrations is one of the most valuable potentials for clinical application (Cobs-Rosas et al., 2015) In the literature, modified sugar beet pectin reduced the number of viable HT29 colon cancer cells by 20.7% in 48 h at 1000 μg mL−1 (Maxwell et al., 2016) Low-molecular-weight citrus pectin decreased cell viability of AGS (gastric cancer) and SW-480 (colorectal cancer) cells by 24% and 28%, respectively, at a concentration of 5.0 mg mL−1 for 24 h (Wang, Li, Lu, & Ling, 2016) However, until now, only one study has been reported for glioblastoma model, when Fan et al (2012) evaluated the effect of Panax ginseng (ginseng) pectin on U87 human glioblastoma cells, and no cytotoxicity was observed at any tested concentrations (10–500 μg mL−1) Considering that one of the pivotal pathways to activate cell death is by excessive reactive oxygen species (ROS) levels (Kalyanaraman et al., 2018), the impact of GW and GWP-FP-S on intracellular ROS levels was measured using DCFH-DA probe As illustrated in Fig 7, the ROS levels were significantly increased after treatment with 100–400 μg mL−1 of GW, and 25–400 μg mL−1 of GWP-FP-S, in comparison to untreated U251-MG cells, which suggest that cytotoxic effect of pectin fractions could be related to increased intracellular ROS levels Citrus pectin (CP) and apple pectin (AP) have also been reported to suppress viability in MDA-MB-231, MCF-7 and T47D human breast cancer cells by increasing ROS content, which led to the caspase-dependent apoptosis (Salehi, Behboudi, Kavoosi, & Ardestani, 2018) The Fig Cytotoxic effect of GW and GWP-FP-S pectins U251-MG cells were treated with GW or GWP-FP-S at different concentrations (10–400 μg mL−1) for 48 h The percentage of metabolically active cells were evaluated by MTT assay The values represent the means ± SD of percentage of cells compared with the control (data represent at least three independent experiments, each in quadruplicate) *p < 0.05; **p < 0.01; ***p < 0.001 activity (19.55%) was observed using 50 μg mL−1 of GW (similar to obtained with high concentrations), while half that amount (25 μg mL−1) of GWP-FP-S was necessary to achieve the same effect (inhibition of 20.96% of metabolic human glioblastoma cell activity) GW and GWP-FP-S treatments at 25 μg mL−1 decreased comparable numbers of adherent cells; nevertheless, U251-MG cells treated with Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al was observed in high concentrations (the cytotoxic effect was not statistically different for treatments using more than 25 μg mL−1) However, is it possible to suggest that the major component responsible for the biological activity of the crude GW is purified GWP-FP-S structure, since after purification process, lower concentrations of this homogeneous fraction were sufficient to exert the cytotoxic effect The crude GW fraction was revealed to be a heterogeneous fraction, with a wide range of molar mass distribution and a degree of methylesterification (DM) of 60% It was composed mostly of RG-I portion (65.3%) and presented high extension of the neutral side chains and high amounts of arabinose (54.5%) and galactose (7.6%) (Barbieri et al., 2019) Evidence from the literature suggests that the antitumor activity may be related to the RG-I domain, possibly by adopting a conformation that maximizes the availability of neutral sugar side chains for cellular interaction (Maxwell et al., 2016; Prado et al., 2017; Vayssade et al., 2010) In addition, the importance of arabinose’s presence in the structure has recently been recognized due to its partial influence on the pectin’s effect on cancer cell lines Maxwell et al (2016) observed a reduction of colon cancer cell proliferation—reaching 7–15% of inhibition—after selective removal of arabinose in modified sugar beet pectin In contrast, GWP-FP-S structure, represented by 57.7% HG and lower proportions (42.0%) of branched RG-I (containing 28.5% of Ara and 11.3% of Gal) and de-esterified (due to the employed alkaline pH in the Fehling’s treatment) By the HPSEC-MALLS-RI analysis, it showed as a homogeneous fraction, with low molar mass (29,170 g mol−1) The molar mass of GWP-FP-S was similar to a modified citrus pectin (MCP, 30,000 g mol−1) (Gao et al., 2012) Modified pectins with lower molar mass have been reported as producing more profound antitumor activity than naturally large pectins (Naqash, Masoodi, Rather, Wani, & Gani, 2017; Zhang et al., 2015) These authors showed the MCP as a galectin-3 (gal3) inhibitor (a carbohydrate recognition domain with the ability to modulate important functions for cell survival migration and metastasis) that represents an attractive target for cancer therapy (Vladoiu, Labrie, & St-Pierre, 2014) Another structural characteristic of pectins attributed to the anticancer effect by inhibiting gal3 function is the presence of →4)-β-DGalp-(1→ units on the branched chain in the RG-I (Gunning, Bongaerts, & Morris, 2009; Zhang et al., 2015) This may explain the difference of biological effect of GW and GWP-FP-S observed in lower concentrations (25 μg mL−1 by MTT and ROS assay), where only GWP-FP-S (which contains 1.5 times more galactose than GW) is capable to induce cytotoxicity In order to evaluate the cytotoxic effect of GW and GWP-FP-S fractions on normal cells, NIH 3T3 murine fibroblast cells were exposed to the same treatment conditions as GW and GWP-FP-S (10–400 μg mL−1 for 48 h), and cytotoxic effect was measured by MTT assay Both pectin fractions showed no cytotoxicity for NIH 3T3 cells after 48 h of treatment at all tested concentrations (Fig 8) By contrast, traditional anticancer agents are generally toxic to normal cells, producing severe side effects (Huettemann & Sakka, 2005; Wang, Huang, Sun, & Pan, 2015) Indeed, pectins have been described as a biocompatible and nontoxic biopolymer (Chan et al., 2017; Munarin et al., 2015) In addition, 400 μg mL−1 (or 13.7 μmol L-1) of GWP-FP-S, the highest concentration tested on the human glioblastoma model, inhibited 26.5% of metabolically active glioblastoma cells; in contrast, temozolomide (TMZ), the standard chemotherapy for glioma patients (Bailey et al., 2015), is known to inhibit 24.7% of U251-MG viable cells (also evaluated by MTT assay, on 48 h of treatment) at 100 μmol L−1 (Shen, Hu, & Zheng, 2014) Interestingly, GWP-FP-S concentration is approximately times lower than the necessary TMZ concentration for a similar effect The antitumor activities of polysaccharides can be mediated through three main approaches: direct cytotoxicity, immunoenhancement, and synergistic effects combined with conventional antitumor drugs (Yang et al., 2013) In this context, GW and GWP-FP-S pectin Fig Effect of GW and GWP-FP-S pectins on intracellular ROS levels U251MG cells were treated with GW or GWP-FP-S at different concentrations (25–400 μg mL−1) for 48 h The intracellular ROS levels were evaluated spectrofluorometrically using a DCFH-DA probe U251-MG cells treated with hydrogen peroxide (H2O2) were used as a positive control (400 μM, 30 min) The values represent the means ± SD of percentage of cells compared with the control (data represent at least three independent experiments, each in quadruplicate) *p < 0.05; **p < 0.01; ***p < 0.001 Fig Cytotoxic effect of GW and GWP-FP-S pectins on normal fibroblast cells NIH 3T3 cells were treated with GW or GWP-FP-S at different concentrations (10–400 μg mL−1) for 48 h, and MTT assay was performed The values represent the means ± SD of percentage of metabolically active cells compared with the control (data represent at least three independent experiments, each in quadruplicate) *p < 0.05; **p < 0.01; ***p < 0.001 elevated levels of ROS are reported to increase the vulnerability of cancer cells to oxidative damage of various macromolecules, including proteins, lipids, and DNA, which in turn results in cell death (Wang et al., 2019) Furthermore, it is well established in the literature (Galadari, Rahman, Pallichankandy, & Thayyullathil, 2017; Panieri & Santoro, 2016) that cancer cells have an inherent elevated ROS level compared to normal cells This effect suggests that therapeutic strategies that increase ROS generation may push cancer cells beyond the breaking point, leading to a toxic level, thereby activating ROS-induced cell death pathways This may explain why GW and GWP-FP-S were only cytotoxic on glioblastoma cells (Figs and 6) and showed no cytotoxicity for NIH 3T3 cells (Fig 8) Interesting, only the purified pectin (GWP-FP-S) at 25 μg mL−1 was capable to increase significantly (1.3 fold higher than control) the levels of ROS, corroborating to cytotoxic effects observed by MTT assay (Fig 6) In addition, the effect observed for 400 μg mL−1 of GW (1.71 fold) is achieved in a 4-fold lower concentration of GWP-FP-S (100 μg mL−1) These results indicate that, despite differing chemical structural characteristics between GW and GWP-FP-S, a similar cytotoxic effect Carbohydrate Polymers 224 (2019) 115140 S.d.C Amaral, et al fractions from gabiroba pulp could prove to be effective antitumor candidates The purification and chemical structural characterization are important factors in establishing structure-function relationships According to the literature, the cytotoxic effects of pectins on cancer cell lines may be related to different factors involving their structure, as conformation of the molecule, degree of esterification, ratio of HG / RGI and side chain of these polysaccharides In this work, by comparing the data obtained about the chemical structure and response to cytotoxicity in glioblastoma cells, it may suggests that the antitumor activity of the pectic fractions extracted from gabiroba pulp (GW and GWP-FP-S) is probably related to the presence of →4)-β-D-Galp-(1→ units on the branched chain in the RG-I (Gunning et al., 2009; Zhang et al., 2015) The amount of galactose on the GWP-FP-S structure (1.5 times higher than GW fraction) may explain the difference of cytotoxic effect of in lower concentrations (25 μg mL−1 by MTT and ROS assay), where only GWP-FP-S is capable to induce cytotoxicity Else more, for the first time, a crude (GW) and purified (GWP-FP-S) pectins from gabiroba pulp have demonstrated cytotoxicity in glioblastoma cell lines In terms of purely cytotoxic purposes, crude pectin GW presented similar effects, when compared to the purified GWP-FPS, with the advantages of higher yield and fewer steps in the extraction process, which in turn mean lower cost and time of acquisition Nevertheless, GWP-FP-S requires less concentration to exert this effect and is more suitable to continue further studies on the potential antitumor effect, since it is a homogeneous purified pectin References Alice, C B., Siqueira, N C S., Mentz, L A., Brasil, E., Silva, G A A., & Jose, K F D (1995) Plantas medicinais de uso popular: Atlas Farmacognóstico Canoas: ULBRA59–61 Arbab, A S., Rashid, M H., Angara, K., Borin, T F., Lin, P C., Jain, M., et al (2017) Major challenges and potential microenvironment-targeted therapies in glioblastoma International Journal of Molecular Sciences, 18, 2732–2750 Barbieri, S F., Ruthes, A C., Petkowicz, C L O., Godoy, R C B., Sassaki, G L., SantanaFilho, A., et al (2017) Extraction, purification and structural characterization of a galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg), Myrtaceae family Carbohydrate Polymers, 174, 887–895 Barbieri, S F., Amaral, S C., Ruthes, A C., Petkowicz, C L O., Kerkhoven, N C., Silva, E R A., et al (2019) Pectins from the pulp of gabiroba (Campomanesia xanthocarpa Berg): Structural characterization and rheological behavior Carbohydrate Polymers, 214, 250–258 Barroso, G M (1978) Sistemática das magnoliophytas In G M Barroso (Ed.) 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